Process for producing kiss-1 peptide

ABSTRACT

A KiSS-1 Peptide or a salt thereof can be industrially mass-produced by subjecting a fused protein or peptide in which the KiSS-1 peptide is ligated to the N-terminal of a low-molecular peptide having cysteine at its N-terminal to the cleavage reaction of the peptide linkage on the amino group side of said cysteine residue.

TECHNICAL FIELD

The present invention relates to a method for producing a KiSS-1 peptideor a salt thereof by preparing a fused protein or polypeptide in whichthe KiSS-1 peptide is ligated to the N-terminal of a low-molecularpeptide having cysteine at its N-terminal, and then subjecting saidfused protein or polypeptide to the cleavage reaction of the peptidelinkage.

BACKGROUND ART

In order to produce peptides using a genetic engineering technology, thepeptides expressed in the form of fused proteins is frequently employedsince peptides are susceptible to be cleaved intracellularly. Knownmethods of cleaving the desired peptide from the fused protein include achemical method using bromocyan (Itakura et al., Science, 198, 1056(1977)) or an enzymatic method using Factor-Xa (Nagai et al., Methods inEnzymology, 153, 46 (1987)).

Further, as a method of cleaving the peptide linkage in a protein, it isknown that acylcysteine bond is cleaved by 2-nitro-5-thiocyanobenzoicacid (Chemistry of Protein II of Lectures on Biochemical Experiment 1,edited by Japanese Biochemical Society and published by Tokyo KagakuDozin, pp. 247-250, 1976). However, there has been no disclosure on theexcision of the desired peptide from a protein.

WO00/24890 and WO01/75104 disclose a KiSS-1 peptide or a salt thereof,which is used in the present invention.

WO01/44469 discloses a method for producing a KiSS-1 peptide or a saltthereof, which comprises subjecting a fused protein, a peptide or a saltthereof in which the KiSS-1 peptide is ligated to the N-terminal of aprotein or peptide having cysteine at its N-terminal, to the cleavagereaction of the peptide linkage on the amino group side of said cysteineresidue. The protein or peptide having cysteine at its N-terminalincludes interferons, interleukins, various growth factors such asfibroblast growth factor (aFGF, bFGF), (pro)urokinases, lymphotoxin,Tumor Necrosis Factor (TNF), enzyme proteins such as β-galactosidase,storage proteins, streptoavidin, protein A, protein G, TissuePlasminogen Activator (TPA), or muteins or a part thereof (a fragment).

The prior art method which involves use of bromocyan cannot be appliedto the production of methionine-containing peptides, while the methodhas drawbacks, for example in terms of excision yield.

Therefore, a demand exists for a technology by which the desired proteinor peptide may be efficiently excised from the fused protein orpolypeptide.

DISCLOSURE OF INVENTION

The present inventors have investigated in detail on a method ofefficiently producing a KiSS-1 peptide or a salt thereof, which is anovel physiologically active peptide. As a result, the present inventorshave discovered that a KiSS-1 peptide or a salt thereof can beefficiently produced by preparing a fused protein or polypeptide, inwhich a KiSS-1 peptide is ligated to the N-terminal of a low-molecularpeptide having cysteine at its N-terminal, and then subjecting the fusedprotein or polypeptide to the cleavage reaction of the peptide linkage.

That is, the present invention provides:

-   -   (1) A method for producing a KiSS-1 peptide or a salt thereof,        which comprises subjecting a fused protein, a peptide or a salt        thereof in which the KiSS-1 peptide is ligated to the N-terminal        of a low-molecular peptide having cysteine at its N-terminal to        the cleavage reaction of the peptide linkage on the amino group        side of said cysteine residue;    -   (2) A method for producing a KiSS-1 peptide or a salt thereof,        which comprises cultivating a transformant having a vector        comprising a DNA encoding a fused protein or a peptide in which        the KiSS-1 peptide is ligated to the N-terminal of a        low-molecular peptide having cysteine at its N-terminal to        express the fused protein, the peptide or a salt thereof, and        subjecting the expressed fused protein, the peptide, or the salt        thereof to the cleavage reaction of the peptide linkage on the        amino group side of said cysteine residue;    -   (3) The method according to the above (1) or (2), wherein the        C-terminal of the KiSS-1 peptide is an amide;    -   (4) The method according to the above (1) or (2), wherein the        cleavage reaction is an S-cyanylation reaction followed by an        ammonolysis or a hydrolysis;    -   (5) The method according to the above (1) or (2), wherein the        KiSS-1 peptide is a peptide comprising an amino acid sequence of        SEQ ID NO: 1;    -   (6) The method according to the above (1) or (2), wherein the        KiSS-1 peptide is (i) a peptide having an amino acid sequence of        position 40 to position 54 from the N-terminal in the amino acid        sequence of SEQ ID NO: 1; (ii) a peptide having an amino acid        sequence of position 45 to position 54 from the N-terminal in        the amino acid sequence of SEQ ID NO: 1; (iii) a peptide having        an amino acid sequence of position 46 to position 54 from the        N-terminal in the amino acid sequence of SEQ ID NO: 1; or (iv) a        peptide having an amino acid sequence of position 47 to position        54 from the N-terminal in the amino acid sequence of SEQ ID NO:        1;    -   (7) The method according to the above (1) or (2), wherein the        low-molecular peptide having cysteine at the N-terminal is a        peptide having cycteine at the N-terminal and consisting of        about 10 to about 50 amino acid residues;    -   (8) The method according to the above (1) or (2), wherein the        low-molecular peptide is a partial peptide on the C-terminal        side of a precursor protein comprising the KiSS-1 peptide, and        has an amino acid sequence starting from an amino acid residue        adjacent to the C-terminal amino acid of the KiSS-1 peptide;    -   (9) The method according to the above (1) or (2), wherein the        low-molecular peptide having cysteine at the N-terminal is a        peptide that comprises the amino acid sequence of SEQ ID NO: 3        and has a cysteine residue at the N-terminal;    -   (10) The method according to the above (1) or (2), wherein the        low-molecular peptide having cysteine at the N-terminal is a        peptide that comprises an amino acid sequence of SEQ ID NO: 3        and has cysteine residue at the N-terminal; the KiSS-1 peptide        is a peptide having an amino acid sequence of SEQ ID NO: 1; and        the KiSS-1 peptide to be produced is a peptide having an amino        acid sequence of SEQ ID NO: 1 with an amide form of the        C-terminal;    -   (11) A fused protein, a peptide, or a salt thereof, in which a        KiSS-1 peptide is ligated to the N-terminal of a low-molecular        peptide having cysteine at its N-terminal;    -   (12) The fused protein, the peptide or the salt thereof        according to the above (11), comprising an amino acid sequence        of SEQ ID NO: 5;    -   (13) DNA comprising DNA encoding the fused protein or the        peptide according to the above (11);    -   (14) DNA according to the above (13), having (i) a base sequence        of SEQ ID NO: 6; or (ii) a base sequence of SEQ ID NO: 7;    -   (15) A vector comprising the DNA according to the above (13);    -   (16) A transformant having the vector according to the above        (15); and    -   (17) Escherichia coli MM294 (DE3)/pTC2MetC24-1.3 identified as        FERM BP-7823.

Further, the present invention provides:

-   -   (18) The method according to the above (2), comprising the        following steps (i) to (iv) of:    -   (i) producing a DNA encoding a fused protein or a peptide, in        which a KiSS-1 peptide is ligated to the N-terminal cysteine of        a low-molecular peptide having cysteine at its N-terminal;    -   (ii) producing a vector comprising said DNA;    -   (iii) cultivating a transformant having said vector to express        the fused protein, the peptide or a salt thereof; and    -   (iv) subjecting the expressed fused protein, the peptide, or the        salt thereof to the cleavage reaction of the peptide linkage on        the amino group side of said cysteine residue;    -   (19) a method for producing a target mature peptide or a salt        thereof, which comprises subjecting a fused protein, a peptide        or a salt thereof in which the target mature peptide is ligated        to the N-terminal of a low-molecular peptide having cysteine at        its N-terminal (wherein the low-molecular peptide means a        partial peptide on the C-terminal side of a precursor protein of        the target mature peptide), to the cleavage reaction of the        peptide linkage on the amino group side of said cysteine        residue;    -   (20) A method for producing a target mature peptide or a salt        thereof, which comprises cultivating a transformant having a        vector comprising DNA encoding a fused protein or a peptide in        which the target mature peptide is ligated to the N-terminal of        a low-molecular peptide having cysteine at its N-terminal        (wherein the low-molecular peptide means a partial peptide on        the C-terminal side of a precursor protein of the target mature        peptide) to express the fused protein, the peptide, or a salt        thereof; and subjecting the expressed fused protein, the        peptide, or the salt thereof to the cleavage reaction of the        peptide linkage on the amino group side of said cysteine        residue;    -   (21) The method according to the above (19) or (20), wherein the        cleavage reaction is an S-cyanylation reaction followed by an        ammonolysis or a hydrolysis;    -   (22) The method according to the above (19) or (20), wherein the        target mature peptide is a peptide comprising about 10 to 100        amino acid residues;    -   (23) The method according to the above (19) or (20), wherein the        low-molecular peptide having cysteine at the N-terminal is a        peptide consisting of about 10 to about 50 amino acid residues;    -   (24) A fused protein, a peptide, or a salt thereof, in which a        target mature peptide is ligated to the N-terminal of a        low-molecular peptide having cysteine at its N-terminal (wherein        the low-molecular peptide means a partial peptide on the        C-terminal side of a precursor protein of the target mature        peptide);    -   (25) DNA comprising DNA encoding the fused protein or the        peptide according to the above (24);    -   (26) A vector comprising the DNA according to the above (25);    -   (27) A transformant having the vector according to the above        (26); and    -   (28) The method according to the above (20), comprising the        following steps (i) to (iv) of:    -   (i) producing a DNA encoding a fused protein or a peptide in        which a target mature peptide is ligated to the N-terminal of a        low-molecular peptide having cysteine at its N-terminal (wherein        the low-molecular peptide means a partial peptide on the        C-terminal side of a precursor protein of the target mature        peptide);    -   (ii) producing a vector comprising said DNA;    -   (iii) cultivating a transformant having said vector to express        the fused protein, the peptide, or the salt thereof; and    -   (iv) subjecting the expressed fused protein, the peptide, or the        salt thereof to the cleavage reaction of the peptide linkage on        the amino group side of said cysteine residue.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the DNA fragments used in Example 1;

FIG. 2 shows the schematic diagram of the construction of plasmidpTC2MetC24 obtained in Example 1; and

FIG. 3 shows the schematic diagram of the construction of the plasmidpTC2MetC24 obtained in Example 5.

BEST MODE FOR CARRYING OUT THE INVENTION

The KiSS-1 peptide used in the method of the present invention includes,for example, the human KiSS-1 peptide described in WO00/24890, and themouse or rat KiSS-1 peptide described in WO01/75104.

The human KiSS-1 peptide specifically includes a peptide that contains asequence of position 47 to position 54 from the N-terminal in the aminoacid sequence of SEQ ID NO: 1, and consists of 8 to 54 amino acidresidues, etc.

The “peptide that contains a sequence of position 47 to position 54 fromthe N-terminal in the amino acid sequence of SEQ ID NO: 1, and consistsof 8 to 54 amino acid residues” may be selected from any peptides whichcontain a sequence of position 47 to position 54 from the N-terminal inthe amino acid sequence of SEQ ID NO: 1 and consist of 8 to 54 aminoacid residues, provided that the peptide activity (for example, thebinding activity between the peptide and its receptor, or cellstimulation activity on the cell expressing the receptor caused by thepeptide, etc.) is retained to the substantially same extent.Specifically, the following may be used: (i) a peptide having an aminoacid sequence of SEQ ID NO: 1; and (ii) a peptide containing a sequenceof position 47 to position 54 from the N-terminal in the amino acidsequence SEQ ID NO: 1 at the C-terminal, and consisting of 8 to 15 aminoacid residues.

More specifically, the following may be used as the human KiSS-1peptide: (i) a peptide having an amino acid sequence of SEQ ID NO: 1;(ii) a peptide containing a sequence of position 40 to position 54 fromthe N-terminal in the amino acid sequence SEQ ID NO: 1; (iii) a peptidecontaining a sequence of position 45 to position 54 from the N-terminalin the amino acid sequence of SEQ ID NO: 1; (iv) a peptide containing asequence of position 46 to position 54 from the N-terminal in the aminoacid sequence SEQ ID NO: 1; and (v) a peptide containing a sequence ofposition 47 to position 54 from the N-terminal in the amino acidsequence SEQ ID NO: 1.

As the mouse KiSS-1 peptide (A), for example, (i) a peptide containing asequence of position 134 to position 141 from the N-terminal in theamino acid sequence of SEQ ID NO: 16 and consisting of 8 to 52 aminoacid residues, may be used. Specifically, the following may be used asthe mouse KiSS-1 peptide: (i) a peptide containing a sequence ofposition 90 to position 141 from the N-terminal in the amino acidsequence of SEQ ID NO: 16; (ii) a peptide containing a sequence ofposition 132 to position 141 from the N-terminal in the amino acidsequence SEQ ID NO: 16; and (iii) a peptide containing a sequence ofposition 127 to position 141 from the N-terminal in the amino acidsequence of SEQ ID NO: 16.

As the mouse KiSS-1 peptide (B), for example, a peptide containing asequence of position 138 to position 145 from the N-terminal in theamino acid sequence of SEQ ID NO: 17 and consisting of 8 to 52 aminoacid residues, may be used. Specifically, a peptide containing asequence of position 94 to position 145 from the N-terminal in the aminoacid sequence of SEQ ID NO: 17, may be used.

As the rat KiSS-1 peptide, for example, a peptide containing a sequenceof position 112 to position 119 from the N-terminal in the amino acidsequence SEQ ID NO: 18 and consisting of 8 to 52 amino acid residues,may be used. Specifically, the following may be used as the rat KiSS-1peptide: (i) a peptide containing a sequence of position 68 to position119 from the N-terminal in the amino acid sequence of SEQ ID NO: 18;(ii) a peptide containing a sequence of position 110 to position 119from the N-terminal in the amino acid sequence of SEQ ID NO: 18; and(iii) a peptide containing a sequence of position 105 to position 119from the N-terminal in the amino acid sequence of SEQ ID NO: 18.

The above-mentioned KiSS-1 peptides have a ligand activity to thereceptor protein, OT7T175 described in WO00/24890 or WO01/75104.

The peptides of the present specification are designated by theconventional way of describing peptides. That is, the left end is theN-terminal (amino terminal) and the right end is the C-terminal(carboxyl terminal). The C-terminal of the peptide represented by SEQ IDNO: 1 may be amide (—CONH₂), a carboxyl group (—COOH), a carboxylate(—COO—), an alkylamide (—CONHR), or an ester (—COOR). R of the ester oralkylamide includes, for example, a C₁₋₆ alkyl group such as methyl,ethyl, n-propyl, isopropyl, and n-butyl; a C₃₋₈ cycloalkyl group such ascyclopentyl and cyclohexyl; a C₆₋₁₂ aryl group such as phenyl andα-naphthyl; a phenyl-C₁₋₂ alkyl such as benzyl, phenethyl andbenzhydryl; or a C₇₋₁₄ aralkyl group such as an α-naphthyl-C₁₋₂ alkylgroup, e.g., α-naphthylmethyl; and the like. In addition, apivaloyloxymethyl group or the like which is used widely as an ester fororal administration may also be used.

Further, the KiSS-1 peptide includes peptides wherein the amino group ofN-terminal methionine residue of the above peptide is protected with aprotecting group (e.g., C₁₋₆ acyl group such as C₂₋₆ alkanoyl group,e.g., formyl group, acetyl group, etc.); those wherein the N-terminalside is cleaved in vivo and the glutamyl group formed ispyroglutaminated; and those wherein a substituent (e.g., —OH, —SH,—COOH, amino group, imidazole group, indole group, guanidino group,etc.) on the side chains of an amino acid in the molecule of the peptideis protected with an appropriate protecting group (e.g., C₁₋₆ acyl groupsuch as C₂₋₆ alkanoyl group, e.g., formyl group, acetyl group, etc.), orconjugated peptides such as glycopeptides with sugar chains attachedthereto.

Salts of the KiSS-1 peptide of the present invention may be ones withphysiologically acceptable bases (for example, alkali metals, etc.) oracids (organic acids or inorganic acids), preferably physiologicallyacceptable acid addition salts thereof. Examples of such salts includesalts with inorganic acids (e.g., hydrochloric acid, phosphoric acid,hydrobromic acid, and sulfuric acid), salts with organic acids (e.g.,acetic acid, formic acid, propionic acid, fumaric acid, maleic acid,succinic acid, tartaric acid, citric acid, malic acid, oxalic acid,benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

In the method of the present invention, the proteins or peptides havingcysteine at its N-terminal are not specified. If there is no cysteine atthe N-terminal, a protein or a peptide may have cysteine at itsN-terminal according to a publicly known method.

The “low-molecular peptide” in the low-molecular peptide having cysteineat its N-terminal has, for example, about 10 to 50, preferably about 20to 40, more preferably about 20 to 30 amino acid residues. Among others,(i) a partial peptide of the human KiSS-1 peptide precursor having theamino acid sequence of SEQ ID NO: 15 (for example, J. Natl. CancerInst., 88, 1731, 1996; WO98/39448), (ii) a partial peptide of the mouseKiSS-1 peptide precursor (A) having the amino acid sequence of SEQ IDNO: 16 (WO01/75104), (iii) a partial peptide of the mouse KiSS-1 peptideprecursor (B) having the amino acid sequence of SEQ ID NO: 17(WO01/75104), (iv) a partial peptide of the rat KiSS-1 peptide precursorhaving the amino acid sequence of SEQ ID NO: 18 (WO01/75104) and thelike may be used. Among others, a partial peptide on the C-terminal sideof such KiSS-1 peptide precursors is preferred. More preferably, thelow-molecular peptide is a partial peptide on the C-terminal side of theprecursor protein containing the KiSS-1 peptide, and has an amino acidsequence starting from the amino acid residues adjacent to theC-terminal amino acid of the KiSS-1 peptide, etc.

More specifically, the following may be used as the low-molecularpeptide:

-   -   (1) When the KiSS-1 peptide is the human KiSS-1 peptide, a        partial peptide on the C-terminal side of the human KiSS-1        peptide precursor having the amino acid sequence of SEQ ID NO:3,        etc.;    -   (2) When the KiSS-1 peptide is the mouse KiSS-1 peptide (A), a        partial peptide on the C-terminal side of the mouse KiSS-1        peptide precursor containing a sequence of position 142 to        position 152 from the N-terminal in the amino acid sequence of        SEQ ID NO: 16;    -   (3) When the KiSS-1 peptide is the mouse KiSS-1 peptide (B), a        partial peptide on the C-terminal side of the mouse KiSS-1        peptide precursor containing a sequence of position 146 to        position 156 from the N-terminal in the amino acid sequence of        SEQ ID NO: 17; and    -   (4) When the KiSS-1 peptide is the rat KiSS-1 peptide, a partial        peptide on the C-terminal side of the rat KiSS-1 peptide        precursor containing a sequence of position 120 to position 130        from the N-terminal in the amino acid sequence of SEQ ID NO: 18.

In the method of the present invention, cysteine is ligated to theN-terminal of such low-molecular peptides.

The DNA encoding the fused protein (including the fused peptides) usedfor the method of the present invention may be constructed by (1)chemically synthesizing the entire base sequence, or (2) placing a basesequence encoding cysteine on the N-terminal side of a base sequenceencoding a low-molecular peptide and placing a base sequence encoding aKiSS-1 peptide on the N-terminal side. Also, when it is desired toobtain a fragment of said peptide, the DNA may be constructed by (3)substituting the amino acid residue immediately after the desiredfragment with cysteine by, for example, site-directed mutagenesis orother technique.

In the case of the above (1), the desired DNA can be prepared byligation using T4 DNA ligase after synthesis of the entire sequence atonce if it is short or in separate steps if it is long, by the publiclyknown phosphoamidide method, phosphotriester method, diester method orhydrogen phosphonate method.

In the case of the above (2), the desired DNA can be obtained asfollows: The DNA encoding the C-terminal protein is obtained by cleavagefrom chromosome or cDNA with the appropriate restriction enzymes andsubsequent ligation with a vector, or by obtaining a cDNA, which is thencleaved with restriction enzymes so that cysteine is present at itsN-terminal or modified to have cysteine at its N-terminal by ligating asynthetic DNA to the 5′-terminal of the entire protein or partial genethereof. The 5′-terminal is ligated to a DNA encoding the desiredprotein (which DNA may be chemically synthesized or may be cloned froman organism).

Examples of the thus obtained DNA encoding the fused protein include theDNA containing a base sequence (SEQ ID NO: 6 or 7) represented by thefollowing formula: (SEQ ID NO: 2)GGTACTTCTCTGTCTCCGCCGCCGGAATCTTCTGGTTCTCGTCAGCAGCCGGGTCTGTCTGCTCCGCACTCTCGTCAGATCCCGGCTCCGCAGGGTGCTGTTCTGGTTCAGCGTGAAAAAGACCTGCCGAACTACAACTGGAACTCTTTC GGTCTGCGTTTC -TGC orTGT-R (I)

-   -   [wherein R represents the base sequence represented by SEQ ID        NO: 4].

The above formula (I) shows that the base sequence represented by R (SEQID NO: 4) is ligated to the DNA base sequence (SEQ ID NO: 2) encoding apeptide comprising a human KiSS-1 peptide through the base sequenceencoding cysteine (TGC or TGT).

The DNA encoding the human KiSS-1 peptide may also be produced accordingto any publicly known method using DNA represented by the formula (I),DNA encoding the human KiSS-1 peptide precursor containing the aminoacid sequence of SEQ ID NO: 15, or modified DNA thereof (for example, J.Natl. Cancer Inst., 88, 1731, 1996; WO98/39448).

The DNA encoding the mouse KiSS-1 peptide may be produced according toany publicly known method using DNA (SEQ ID NO: 19) encoding a mouseKiSS-1 peptide precursor (A) having the amino acid sequence of SEQ IDNO: 16, or modified DNA thereof (WO01/75104), or DNA (SEQ ID NO: 20)encoding a mouse KiSS-1 peptide precursor (B) having the amino acidsequence of SEQ ID NO: 17, or a modified DNA thereof (WO01/75104).

The DNA encoding the rat KiSS-1 peptide may be produced according to anypublicly known method using DNA (SEQ ID NO: 21) encoding a rat KiSS-1peptide precursor having the amino acid sequence of SEQ ID NO: 18, or amodified DNA thereof (WO01/75104).

A DNA (plasmid) which has ATG at its 5′-terminal, a region encoding thefused protein downstream thereof and a translation termination codonfurther downstream can be produced by chemical synthesis or byprocessing known cDNA of said protein produced by genetic engineering orby processing the chromosome-derived DNA of said protein.

In the present invention, the DNA encoding a fused protein or a peptidein which a KiSS-1 peptide is ligated to the N-terminal of thelow-molecular peptide having cysteine at its N-terminal, can be modifiedto DNA encoding the desired mutein by using the conventional DNAtechnology, for example, site-directed mutagenesis.

Site-directed mutagenesis is well-known, and it is described in GeneticEngineering, Lather, R. F. and Lecoq, J. P., Academic Press, pp. 31 to50 (1983). Mutagenesis directed to oligonucleotide is described inGenetic Engineering: Principles and Methods, Smith, M. and Gillam, S.,Plenum Press, Vol. 3, pp. 1 to 32 (1981).

Examples of the plasmid used as a vector to produce a plasmid carryingDNA having a region coded for said fused protein include Escherichiacoli-derived plasmids such as pBR322 [Gene, 2, 95 (1977)], pBR313 [Gene,2, 75 (1977)], pBR324, pBR325 [Gene, 4, 124 (1978)], pBR327, pBR328[Gene, 9, 287 (1980)], pBR329 [Gene, 17, 79 (1982)], pKY2289 [Gene, 3, 1(1978)], pKY2700 [Seikagaku, 52, 770 (1980)], pACYC177, pACYC184[Journal of Bacteriology, 134, 1141 (1978)], pRK248, pRK646, pDF[Methods in Enzymology, 68, 268 (1979)], pUC18, pUC19 [Janisch-Perror etal., Gene, 33, 103 (1985)], or the like. Also useful are λgt vectorsusing λ phage such as λgt-λC [Proc. Nat. Acad. Sci., U.S.A., 71, 4579(1974)], λgt-λB [Proc. Nat. Acad. Sci., U.S.A., 72, 3461 (1975)], λDam[Gene, 1, 255 (1977)], Charon vector [Science, 196, 161 (1977); Journalof Virology, 29, 555 (1979)] and mp vectors using a filamentous phagesuch as mp18 and mp19 [Janisch-Perror et al., Gene, 33, 103 (1985)].

The DNA preferably has a promoter upstream of ATG. Any promoter may beused as long as it is suitable for the host in producing a transformant.

Examples of such promoters include the trp promoter, lac promoter, rec Apromoter, λPL promoter, lpp promoter and T7 promoter for Escherichiacoli, SPO1 promoter, SPO2 promoter and penP promoter for Bacillussubtilis, PHO5 promoter, PGK promoter, GAP promoter and ADH promoter forSaccharomyces cerevisiae and the SV40-derived promoter for animal cells.The SD (Shine and Dalgarno) sequence may be inserted downstream of thepromoter as necessary.

When using the T7 promoter system, the promoter may be any of the 17promoters found on the T7 DNA [J. L. Oakley et al., Proc. Natl. Acad.Sci., U.S.A., 74, 4266-4270 (1977); M. D. Rosa, Cell, 16, 815-825(1979); N. Panayotatos et al., Nature, 280, 35 (1979); J. J. Dunn etal., J. Mol. Biol., 166, 477-535 (1983)]. The φ10 promoter [A. H.Rosenberg et al., Gene, 56, 125-135 (1987)] is most preferable.

Any transcription terminator can be used, as long as it functions inEscherichia coli systems, but the Tφ terminator [F. W. Studier et al.,J. Mol. Biol., 189, 113-130 (1986)] is preferred.

The T7 RNA polymerase gene is exemplified by the T7 gene [F. W. Studieret al., J. Mol. Biol., 189, 113-130 (1986)].

The vector is preferably constructed by inserting the T7 promoter and T7terminator into the above-mentioned vector. Examples of such vectorsinclude pET-1, pET-2, pET-3, pET-4 and pET-5 [A. H. Rosenberg, Gene, 56,125-135 (1987)], and pTB960-2 (EP-A-499990)), but pTB960-2 is preferablyused.

The transformant of the present invention can be produced bytransforming a host with an expression plasmid obtained as describedabove using a publicly known method [e.g., Cohen S. N. et al.,Proceedings of National Academy of Science, U.S.A., 69, 2110 (1972)].

Examples of the microorganism host to be transformed include bacteriabelonging to the genus Escherichia, bacteria belonging to the genusBacillus, yeasts, and animal cells.

Examples of the bacteria belonging to the genus Escherichia includeEscherichia coli (E. coli), specifically Escherichia coli K12DH1[Proceedings of National Academy of Science, U.S.A., 60, 160 (1968)],JM-103 [Nucleic Acids Research, 9, 309 (1981)], JA221 [Journal ofMolecular Biology, 120, 517 (1978)], HB101 [Journal of MolecularBiology, 41, 459 (1969)], C600 [Genetics, 39, 440 (1954)], N4830 [Cell,25, 713 (1981)], K-12MM294 [Proceedings of National Academy of Science,U.S.A., 73, 4174 (1976)] and BL21.

Examples of the bacteria belonging to the genus Bacillus includeBacillus subtilis, specifically Bacillus subtilis MI114 [Gene, 24, 255(1983)] and 207-21 [Journal of Biochemistry, 95, 87 (1984)].

Examples of the yeasts include Saccharomyces cerevisiae, specificallySaccharomyces cerevisiae AH22 [Proceedings of National Academy ofScience, U.S.A., 75, 1929 (1978)], XSB52-23C [Proceedings of NationalAcademy of Science, U.S.A., 77, 2173 (1980)], BH-641A (ATCC 28339),20B-12 [Genetics, 85, 23 (1976)] and GM3C-2 (Proceedings of NationalAcademy of Science, U.S.A., 78, 2258 (1981).

Examples of the animal cells include simian cells COS-7 [Cell, 23, 175(1981)], Vero [Japanese Journal of Clinical Medicine, 21, 1209 (1963)],Chinese hamster cells CHO [Journal of Experimental Medicine, 108, 945(1985)], mouse L cells [Journal of National Cancer Institute, 4, 165(1943)], human FL cells [Proceedings of the Society for ExperimentalBiology and Medicine, 94, 532 (1957)] and hamster C cells.

When using a T7 promoter system, any host can be used to obtain thedesired transformant, as long as it is an Escherichia coli straincapable of incorporating the T7 RNA polymerase gene (T7 gene 1) [F. W.Studier et al., J. Mol. Biol., 189, 113-130 (1986)]. Examples of suchstrains are MM294, DH-1, C600, JM109, BL21 or an Escherichia coli strainwith another plasmid with the T7 RNA polymerase gene (T7 gene 1). It ispreferable to use the MM294 strain or BL21 strain resulting fromlysogenization of a λ phage incorporating the T7 gene 1. In this case,the lac promoter is used as promoter for the T7 gene 1, which inducesexpression in the presence of isopropyl-1-thio-β-D-galactopyranoside(which is abbreviated to IPTG).

The transformation of Escherichia bacteria can be carried out using aknown method such as that disclosed in Proceedings of National Academyof Science, U.S.A., 69, 2110 (1972), and Gene, vol. 17, 107 (1982).

The transformation of the host of the genus Bacillus, can be carried outusing a known method such as that disclosed in Molecular & GeneralGenetics, 168, 111 (1979).

The transformation of the host of yeast, can be carried out using aknown method such as that disclosed in Proc. Natl. Acad. Sci. USA, 75,1929 (1978).

The transformation of the host of an animal cell can be carried outusing a known method such as that disclosed in Virology, 52, 456 (1973).

The fused protein can be produced by cultivating the above-mentionedtransformant in a cultured medium and then harvesting the produced fusedprotein.

It is desirable that the pH of medium be about 6 to 8.

An example of a medium for cultivating the bacteria of the genusEscherichia is the M9 medium containing glucose and casamino acids[Miller, Journal of Experiments in Molecular Genetics, 431-433, ColdSpring Harbor Laboratory, New York (1972)]. A chemical substance such as3β-indolylacrylic acid and isopropyl-β-D-thiogalactopyranoside (IPTG)may be added thereto, when it is necessary to increase promoterefficiency.

When the host is a bacterium of the genus Escherichia, cultivation isnormally carried out at about 15 to 43° C. for about 3 to 24 hours, andaeration and/or agitation may also be performed, if necessary.

When the host is a bacterium of the genus Bacillus, cultivation isnormally carried out at about 30 to 40° C. for about 6 to 24 hours, andaeration and/or agitation may also be performed, if necessary.

As a medium for the cultivation of transformants whose host is a yeast,Burkholder's minimum medium may be used [Bostian, K. L. et al., Proc.Natl. Acad. Sci. USA, 77, 4505 (1980)]. It is preferable that the pH ofthe medium be adjusted to about 5 to 8. Cultivation is normally carriedout at about 20 to 35° C. for about 24 to 72 hours, and aeration and/oragitation may also be performed, if necessary.

Media for the cultivation of transformants whose host is an animal cellinclude MEM media containing about 0.2 to 20%, preferably about 5 to 20%fetal bovine serum [Science, 122, 501 (1952)], DMEM medium [Virology, 8,396 (1959)], RPMI1640 medium [Journal of the American MedicalAssociation, 199, 519 (1967)] and 199 medium [Proceeding of the Societyfor the Biological Medicine, 73, 1 (1950)]. It is preferable that pH beabout 6 to 8. Cultivation is normally carried out at about 30 to 40° C.for about 15 to 60 hours, and aeration and/or agitation may beperformed, if necessary.

The fused protein can be produced by cultivating the above-mentionedtransformant, to produce and accumulate the fused protein in a culturedmedium and then harvesting it.

Examples of media include the M9 medium containing glucose and casaminoacids [Miller, Journal of Experiments in Molecular Genetics, 431-433,Cold Spring Harbor Laboratory, New York (1972)] and 2×YT medium[Messing, Methods in Enzymology, 101, 20 (1983)] and LB medium.

Cultivation is normally carried out at about 15 to 43° C. for about 3 to24 hours, and aeration and/or agitation may also be performed, ifnecessary.

When using a recombinant vector having both a λcIts repressor and anexpression vector carrying a λPL-promoter, it is preferable to carry outcultivation of the transformant at a temperature between about 15 and36° C., preferably about 30 and 36° C. and inactivate the λcItsrepressor at a temperature between about 37 and 42° C. Also, to increaserecA promoter efficiency, i.e., to lower the recA gene expressionsuppressive function, a drug such as mitomycin C or nalidixic acid maybe added, ultraviolet irradiation may be employed, or pH of the mediummay be changed t6 alkali, if necessary.

When using a T7 promoter system, (1) IPTG is added to express the T7gene (RNA polymerase gene) ligated downstream from the lac promoter; or(2) the temperature of the culture medium may be elevated in expressingthe T7 gene (RNA polymerase gene) ligated downstream of theλP_(L)-promoter to specifically activate the T7 promoter via theresulting T7 phage RNA polymerase 1.

After cultivation, cells are collected by a known method and suspendedin a buffer, after which they are disrupted by protein denaturanttreatment, ultrasonic treatment, enzymatic treatment using lysozyme,glass bead treatment, French press treatment, freeze-thawing or otherprocess, followed by centrifugation or other known methods, to yield asupernatant.

From the supernatant thus obtained, the fused protein can be isolated inaccordance with known methods of protein purification. For example, gelfiltration, ion exchange chromatography, adsorption chromatography, highperformance liquid chromatography, affinity chromatography, hydrophobicchromatography and electrophoresis can be used in appropriatecombination. The fused protein may be subjected to the subsequentreaction process without purification or in a partially purified state.

The fused protein or peptide thus obtained is then subjected to areaction for cleaving the peptide linkage on the amino group side of thecysteine residue. The cleavage reaction may be, for example, performedby S-cyanylation reaction followed by hydrolysis. If an amide form ofKiSS-1 peptide or a salt thereof is desired as the final product, thecleavage reaction may be performed by the S-cyanylation followed byammonolysis. The S-cyanylation reaction is carried out by reacting anS-cyanylation reagent with the starting compounds.

Examples of S-cyanylation reagents include 2-nitro-5-thiocyanobenzoicacid (NTCB), 1-cyano-4-dimethylaminopyridium salt (DMAP-CN) and CN⁻ ion.The amount of S-cyanylation reagent is about 2 to 50 times, preferablyabout 5 to 10 times the total amount of all thiol groups.

Reaction temperature may be set at any level in the range from about 0to 80° C., preferably between about 0 and 50° C. Any buffer can be usedas a solvent, as long as it does not react with the S-cyanylationreagent. Examples of such buffers include Tris-HCl buffer, Tris-acetatebuffer, phosphate buffer and borate buffer. An organic solvent may bepresent, as long as it does not react with the S-cyanylation reagent.

The reaction may be normally carried out at a pH of 1 to 12.

Particularly when using NTCB, a pH range of from 7 to 10 is preferred.When using DMAP-CN, a pH range of from 2 to 7 is preferred, in order toavoid S—S exchange reaction. The reaction mixture may contain adenaturant such as guanidine hydrochloride.

Reaction for cleaving by hydrolysis or ammonolysis can be achieved, forexample, by alkali treatment.

The alkali treatment is carried out by adjusting a pH of the aqueoussolution containing the starting compound to 7 to 14.

The pH can be adjusted by adding an appropriate amount of a solution ofammonia, sodium hydroxide, amino compound, trizma base(tris[hydroxymethyl]-aminomethane), disodium phosphate, potassiumhydroxide or barium hydroxide, to the aqueous solution containing thestarting compound. In particular, ammonia, etc. is preferable.

The concentration of said solution are, for ammonia or amino compoundabout 0.01 to 15 N, preferably about 0.1 N to 3 N; for sodium hydroxideabout 0.01 to 2 N, preferably about 0.05 N to 1 N; for trizma base about1 mM to 1 M, preferably about 20 mM to 200 mM; for disodium phosphateabout 1 mM to 1 M, preferably about 10 mM to 100 mM; for potassiumhydroxide about 0.01 to 4 N, preferably about 0.1 to 2 N. The reactiontemperature may be between about −20 to 80° C., preferably between about−10 to 50° C.

Reaction times are preferably as follows: for S-cyanylation about 10 to60 minutes, preferably about 15 to 30 minutes; for hydrolysis about 5minutes to 100 hours, preferably about 10 minutes to 15 hours; forammonolysis about 5 minutes to 24 hours, preferably about 10 to 180minutes.

Examples of the amino compound include a compound represented by theformula R¹—(NR²)—H wherein R¹ and R² may be the same or different andrepresent (i) a hydrogen atom; (ii) a C₁₋₂₀ alkyl group, a C₃₋₈cycloalkyl group, a C₆₋₁₄ aryl group, or a C₆₋₁₄ aryl-C₁₋₃ alkyl group(these may have no substituent, or may have 1 to 3 amino groups,hydroxyl groups and the like, on a carbon atom); (iii) an optionallysubstituted amino group; and (iv) a hydroxyl group or a C₁₋₆ alkoxygroup.

The reaction shown in FIG. 1 is considered to occur by S-cyanylation,and ammonolysis or hydrolysis as described above.

As described above, the C-terminal of the KiSS-1 peptide obtainable by aproduction method of the present invention may be amide (—CONH₂),carboxyl group (—COOH), carboxylate (—COO—), alkylamide (—CONHR), orester (—COOR). Amide, carboxyl group or alkylamide is preferable, and inparticular, amide or alkylamide is suitable. Specifically, theC-terminal of the KiSS-1 peptide obtainable by the producing method ofthe present invention may be —CO—X shown in FIG. 1, wherein X representsR¹—(NR²)— (R¹ and R² are the same as defined above) or OH.

Examples of the C₁₋₂₀ alkyl group include methyl, ethyl, propyl,isopropyl, butyl, sec-butyl, pentyl, isopentyl, neopentyl,1-ethylpentyl, hexyl, isohexyl, heptyl, octyl, nonanyl, decanyl,undecanyl, dodecanyl, tetradecanyl, pentadecanyl, hexadecanyl,heptadecanyl, octadecanyl, nonadecanyl, and eicosanyl.

Examples of the C₃₋₈ cycloalkyl group include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

Examples of the C₆₋₁₄₋aryl group include phenyl, naphthyl, anthryl,phenanthryl, and acenaphthylenyl.

Examples of the C₆₋₁₄ aryl-C₁₋₃ alkyl group include benzyl, phenethyl,3-phenylpropyl, (1-naphthyl)methyl, and (2-naphthyl)methyl.

Examples of the C₁₋₆ alkoxy group include methoxy, ethoxy, propoxy,butoxy, pentyloxy, and hexyloxy.

As the substituents of the optionally substituted amino group in theabove (iii), an amino acid and a peptide comprising 2 to 10 amino acidsare exemplified.

Examples of the amino acid include L- or D-isomer of Ala Arg, Asp, Asn,Glu, Gln, Gly, His, Ile, Met, Leu, Phe, Pro, Ser, Thr, Trp, Tyr, andVal.

Examples of the peptide include HD-Leu-Leu-Arg-Pro-NH—C₂H₅, andH-Val-Ala-Leu-D-Ala-Ala-Pro-Leu-Ala-Pro-Arg-OH.

Among others, it is preferred that R² be a hydrogen atom, and R¹ be ahydrogen atom or a C₁₋₂₀ alkyl group.

When using ammonia or an amino compound in the ammonolysis reaction, thecorresponding amide form is obtained.

The desired peptide obtained through cleavage may be isolated inaccordance with known methods of protein purification. For example, gelfiltration, ion exchange chromatography, high performance liquidchromatography, affinity chromatography, hydrophobic chromatography,thin layer chromatography and electrophoresis can be used in appropriatecombination.

The KiSS-1 peptide or a salt thereof, thus obtained may also be isolatedand purified from the reaction solution by a well-known purificationtechnique, for example, extraction, salting out, distribution,recrystallization, or chromatography. Preferable examples of thepurification technique include ion-exchange chromatography through, forexample, SP-Sepharose (Pharmacia Biotech, Co., Ltd.), DEAE-5PW (TosohCorporation), or SP-5PW (Tosoh Corporation), etc.

The KiSS-1 peptide or the salt thereof obtained can be powdered bylyophilization, if necessary. In lyophilization, a stabilizer such assorbitol, mannitol, dextrose, maltose, trehalose or glycerol may beadded.

The KiSS-1 peptide or a salt thereof produced by the method of thepresent invention can be mixed with sterile water, human serum albumin(HSA), physiological saline and other known physiologically acceptablecarriers, and can be administered parenterally or topically to amammalian (e.g. human). It can be administered parenterally byintravenous, intramuscular or other means at a daily dose of about 0.01mg to 50 mg, preferably about 0.1 mg to 10 mg per one person.

Preparations containing the KiSS-1 peptide or a salt thereof produced bythe method of the present invention may contain salts, diluents,adjuvants and other carriers, buffers, binders, surfactants,preservatives and other physiologically acceptable active ingredients.Parenteral preparations are supplied as sterile water solutions, ampulescontaining a suspension in a physiologically acceptable solvent orampules containing a sterile powder normally obtained by lyophilizing apeptide solution which can be freshly prepared in dilution with aphysiologically acceptable diluent for each use.

The KiSS-1 peptide or a salt thereof produced by the method of thepresent invention has an activity of inhibiting cancer metastasis, andtherefore, is useful as a prophylactic or therapeutic drug for all kindsof cancers (for example, lung cancer, stomach cancer, liver cancer,pancreatic cancer, colorectal cancer, rectal cancer, colon cancer,prostate cancer, ovarian cancer, uterine cancer, or breast cancer,etc.).

In addition, the KiSS-1 peptide or a salt thereof has an activity ofcontrolling placental function, and therefore, is useful as aprophylactic or therapeutic drug for choriocarcinoma, vesicular mole,invasive mole, miscarriage, fetal maldevelopment, saccharometabolicdisorder, lipid metabolic disorder, or induction of delivery.

Also, in the method of the present invention, the desired mature peptidecan be produced in a similar manner, by substituting KiSS-1 peptide withthe other desired mature peptide, and further substituting alow-molecular peptide having cysteine at its N-terminal with thecorresponding partial peptide on the C-terminal side of the precursorprotein of the desired mature peptide.

Examples of the other desired mature peptide include a peptide havingamino acid residues of about 10 to 200, preferably about 10 to 100, etc.

Examples of the partial peptide on the C-terminal side of the precursorprotein of the desired mature peptide include a partial peptide on theC-terminal side of a precursor protein containing the desired maturepeptide, having an amino acid sequence starting from an amino acidresidue adjacent to the C-terminal amino acid residue of the desiredmature peptide, etc.

In addition, examples of the partial peptide on the C-terminal side ofthe precursor protein of the desired mature peptide include a partialpeptide having about 10 to 50, preferably about 20 to 40, morepreferably about 20 to 30 amino acid residues on the C-terminal side ofa precursor protein containing the desired mature peptide.

Abbreviations for amino acids, peptides, protective groups, activegroups and other materials used in the present specification andattached drawings are based on abbreviations specified by the IUPAC-IUB(Commission on Biochemical Nomenclature) or abbreviations in common usein relevant fields, examples of which are given below. In addition, forthe amino acids that may have the optical isomers, L-form is presentedunless otherwise indicated.

-   -   DNA: Deoxyribonucleic acid    -   A: Adenine    -   T: Thymine    -   G: Guanine    -   C: Cytosine    -   RNA: Ribonucleic acid    -   EDTA: Ethylenediaminetetraacetic acid    -   Gly: Glycine    -   Ala: Alanine    -   Val: Valine    -   Leu: Leucine    -   Ile: Isoleucine    -   Ser: Serine    -   Thr: Threonine    -   Met: Methionine    -   Glu: Glutamic acid    -   Asp: Aspartic acid    -   Lys: Lysine    -   Arg: Arginine    -   His: Histidine    -   Phe: Phenylalanine    -   Tyr: Tyrosine    -   Trp: Tryptophan    -   Pro: Proline    -   Asn: Asparagine    -   Gln: Glutamine    -   Cys: Cysteine    -   Asx: Asparagine or aspartic acid    -   Glx: Glutamine or glutamic acid    -   ATP: Adenosine triphosphate

The sequence identification numbers in the sequence listing of thespecification indicates the following sequence, respectively.

[SEQ ID NO: 1]

This sequence shows the amino acid sequence of the human KiSS-1 peptide.

[SEQ ID NO: 2]

This sequence shows the base sequence of the DNA encoding the humanKiSS-1 peptide.

[SEQ ID NO: 3]

This sequence shows the amino acid sequence of the low-molecularpeptide.

[SEQ ID NO: 4]

This sequence shows the base sequence of the DNA encoding thelow-molecular peptide.

[SEQ ID NO: 5]

This sequence shows the amino acid sequence of the fused protein.

[SEQ ID NO: 6]

This sequence shows the base sequence 1 of the DNA fragment encoding thefused protein represented by formula (I).

[SEQ ID NO: 7]

This sequence shows the base sequence 2 of the DNA fragment encoding thefused protein represented by formula (I).

[SEQ ID NO: 8]

This sequence shows the base sequence of an oligomer used for preparingthe structural gene of the KiSS-1 peptide in Example 1.

[SEQ ID NO: 9]

This sequence shows the base sequence of the oligomer used for preparingthe structural gene of the KiSS-1 peptide in Example 1.

[SEQ ID NO: 10]

This sequence shows the base sequence of the oligomer used for preparingthe structural gene of the KiSS-1 peptide in Example 1.

[SEQ ID NO: 11]

This sequence shows the base sequence of the oligomer used for preparingthe structural gene of the KiSS-1 peptide in Example 1.

[SEQ ID NO: 12]

This sequence shows the base sequence of the DNA fragment obtained inExample 1.

[SEQ ID NO: 13]

This sequence shows the base sequence of the DNA fragment obtained inExample 1.

[SEQ ID NO: 14]

This sequence shows the base sequence of the DNA fragment obtained inExample 1.

[SEQ ID NO: 15]

This sequence shows the amino acid sequence of the KiSS-1 peptideprecursor.

[SEQ ID NO: 16]

This sequence shows the amino acid sequence of the mouse KiSS-1 peptideprecursor (A).

[SEQ ID NO: 17]

This sequence shows the amino acid sequence of the mouse KiSS-1 peptideprecursor (B).

[SEQ ID NO: 18]

This sequence shows the amino acid sequence of the rat KiSS-1 peptide.

[SEQ ID NO: 19]

This sequence shows the base sequence of the DNA encoding the mouseKiSS-1 peptide precursor (A).

[SEQ ID NO: 20]

This sequence shows the base sequence of the DNA encoding the mouseKiSS-1 peptide precursor (B).

[SEQ ID NO: 21]

This sequence shows the base sequence of the DNA encoding the rat KiSS-1peptide.

The transformant Escherichia coli MM294 (DE3)/pTC2MetC24-1.3 obtained inExample 1 below has been at the International Patent Organism Depositary(IPOD) of the National Institute of the Advanced Industrial Science andTechnology (AIST) Tsukuba Central, 6, 1-1-1 Higashi, Tsukuba, Ibaraki,Japan (Zip code: 305-8566) under the Accession Number FERM BP-7823 sinceDec. 10, 2001 and has been deposited at the Institute for Fermentation,Osaka (IFO), Japan under the accession number IFO 16717 since Oct. 24,2001.

EXAMPLES

The present invention will be described in more detail below withreference to the following examples, which are merely illustrative, andnot intended to limit the present invention.

Example 1

(1) Production of DNA Encoding a KiSS-1 Peptide to Which 24 Amino Acidsare Added at its C-Terminal

(a) Synthesis of DNA Fragments

The gene of the KiSS-1 peptide to which 24 amino acid residues are addedto its C-terminal was prepared using four DNA fragments shown in FIG. 1(#1, #2; 5′-terminal phosphorylated, #3; 5′-terminal phosphorylated, and#4: products of Kikotech).

(b) Ligation of the DNA Fragments for Adding at the C-Terminal

The DNA fragments #1 to #4 obtained in the above (a) were combined to atotal volume of 40 μl. This mixture solution was maintained at 65° C.for 10 minutes and then gradually cooled to room temperature to effectannealing. The ligation reaction was conducted using T4 DNA Ligase(Takara Shuzo) to 10 μl of the annealing solution. Thus, 2 μl of 10×concentration of Ligation buffer and 1 μl of T4 DNA Ligase (350 units)were added to 10 μl of the annealing solution, and after thoroughmixing, the ligation reaction was carried out at 16° C. for 17 hours.Then, heat treatment was conducted at 65° C. for 5 minutes. The DNAfragments thus obtained was phosphorylated using T4 polynucleotidekinase (Takara Shuzo). Then, 96 bp DNA fragment (SEQ ID NO: 12) wasextracted by 1.8% low melting point agarose gel electrophoresis using anELUTIP Minicolumn (Schleicher and Schuell), and dissolved in 20 μl of TEbuffer, which was used in the next (c) step.

(c) Ligation of KiSS-1 Gene and the DNA Fragments for Adding at itsC-Terminal.

pTFC-KiSS-1, an expression vector for KiSS-1 peptide, was digested withNdeI (Takara Shuzo) and XmnI (NEB) at 37° C. for 2 hours, and 149 bp DNAfragment (SEQ ID NO: 13) was extracted by 1.5% low melting point agarosegel electrophoresis using an ELUTIP Minicolumn (Schleicher and Schuell),and dissolved in 20 μl of TE buffer solution. This DNA fragment and theDNA fragment obtained in the above (b) were subjected to ligation. To 20μl of 149 bp DNA solution and 10 μl of 96 bp DNA solution were added 3.5μl of 10× concentration of Ligation buffer and 1.5 μl of T4 DNA Ligase(525 units), and after thorough mixing, the ligation reaction wasconducted at 16° C. for 17 hours. After conducting ligation, heattreatment was carried out at 65° C. for 5 minutes. After conductingdigestion with NdeI and BamHI for 1 hour using this ligation solution in350 μl of a total volume, a DNA fragment (SEQ ID NO: 14) was recoveredby ethanol precipitation. This was used in the next (d) step.

(d) Construction of the Expression Vector for KiSS-1 Peptide to Which 24Amino Acids are Added at its C-Terminal (FIG. 2).

The expression vector pTCII was digested with NdeI and BamHI (TakaraShuzo) at 37° C. for 1 hour. A 4.6 kb DNA fragment was extracted by 1%agarose gel electrophoresis using QIAquick Gel Extraction Kit (Qiagen),and dissolved in 25 μl of TE buffer. This NdeI-BamHI fragment of pTCIIand the DNA fragment obtained in the above (c) were subjected toligation reaction using T4 DNA Ligase (Takara Shuzo).

Competent cells of Escherichia coli JM109 (Takara Shuzo) weretransformed using 10 μl of this reaction solution, then sowed onto LBagar medium containing 10 μg/ml of tetracycline and cultured overnightat 37° C. A tetracycline-resistant colony thus formed was selected. Thistransformant was cultured overnight in LB medium and a plasmidpTC2MetC24 was prepared using QIAprep8 Miniprep Kit (Qiagen). The basesequence of KiSS-1 peptide (having 24 amino acids at its C-terminal)structural gene portion of said plasmid was confirmed using an AppliedBiosystems model 3100 DNA sequencer. The plasmid pTC2MetC24 was used totransform the E. coli MM294 (DE3), and thus KiSS-1 protein (having 24amino acids at its C-terminal) expression strain, MM294(DE3)/pTC2MetC24-1.3, was obtained.

(e) Production of KiSS-1 Peptide to Which 24 Amino Acids are Added atits C-Terminal.

The MM294(DE3)/pTC2MetC24-1.3 was cultured with shaking in a 2-literflask at 37° C. for 8 hours using 1 L of LB medium (1% peptone, 0.5%yeast extract, 0.5% sodium chloride) containing 5.0 mg/L oftetracycline. The culture obtained was transferred to a 50-L fermentercharged with 19 L of a main fermentation medium (1.68% sodiummonohydrogen phosphate, 0.3% potassium dihydrogen phosphate, 0.1%ammonium chloride, 0.05% sodium chloride, 0.025% magnesium sulfate,0.02% antifoam, 0.00025% ferrous sulfate, 0.0005% thiaminehydrochloride, 1.5% glucose, 1.5% casamino acids), and cultivation underaeration and agitation was started at 30° C. When the turbidity of theculture arrived at about 500 Klett units,isopropyl-β-D-thiogalactopyranoside was added to a final concentrationof 12 mg/L, and cultivation was carried out for further 6 hours. Aftercompletion of the cultivation, the culture fluid was centrifuged to giveabout 430 g of wet bacterial cells, which were frozen stored at −80° C.

Example 2

To 70 g of the bacterial cells obtained in Example 1 was added 210 ml ofa solution comprising 7 M guanidine hydrochloride, 100 mM Tris bufferand 1 mM EDTA (pH 8.0), and the mixture was dissolved under stirring andcentrifuged (8,000 rpm, 60 minutes). The supernatant was diluted in a4.2 L solution comprising 50 mM Tris buffer, 0.2 M arginine (pH 8.0), 1mM reduced glutathione and 0.1 mM oxidized glutathione, and stoodovernight in a low-temperature room. This solution was diluted 4 timeswith 1 M urea solution, adjusted to pH 6.0 with acetic acid, and appliedto a SP-Toyopearl 550C column (5 cm ID×20 cm L, Tosoh) equilibrated with50 mM MES-NaOH buffer (pH 6.0), at a flow rate of 750 mL/hour, foradsorption. The column was washed with 50 mM MES-NaOH buffer+0.2 M NaCl(pH 6.0) and then elution was carried out with 50 mM MES-NaOH buffer+0.5M NaCl (pH 6.0). This eluate was diluted 3 times with distilled water,was applied to a SP-5PW column (21.5 mm ID×150 mm L, Tosoh) equilibratedwith 50 mM MES-NaOH buffer (pH 6.0), at a flow rate of 5 mL/minute, foradsorption, and then stepwise gradient elution was carried out with 0 to80% B (B=1 M NaCl+50 mM MES-NaOH buffer, pH 6.0) to pool KiSS-1-24 aminoacid adduct fraction. This eluted fraction was applied to a C4P-50column (21.5 mm ID×300 mm L, Showa Denko) equilibrated with 0.1%trifluoroacetic acid, for adsorption, and stepwise gradient elution wascarried out with 20 to 70% B (B=0.1% trifluoroacetic acid+80%acetonitrile) at a flow rate of 5 mL/minute to give KiSS-1-24 amino acidadduct fraction. This fraction obtained was lyophilized to give alyophilizate powder of KiSS-1-24 amino acid adduct fraction. Thelyophilized powder was dissolved in a solution comprising 0.1 M aceticacid and 6 M urea. Then, about 1.5 mg of DMAP-CN(1-cyano-4-dimethylaminopyridinium tetrafluoroborate) was added thissolution, and the reaction was allowed to proceed at room temperaturefor 15 minutes. After completion of the reaction, the reaction mixturewas applied to a Sephadex G-25 column (2.5 cm ID×50 cm L, Pharmacia)equilibrated with 50 mM monopotassium phosphate and development waseffected using the same 50 mM monopotassium phosphate as used forequilibration at a flow rate of 10 ml/minute to give a fractioncontaining the S-cyanylated KiSS-1 peptide-24 amino acid adduct protein.This eluate was concentrated and desalted using Centriplus (fractionalmolecular weight of 3 kDa:Millipore) to give a desalted solution of theKiSS-1-24 amino acid adduct. Urea was added to this desalted solution toa final concentration of 6 M, 25% aqueous ammonia was further added toan ammonia concentration of 3 M, and the reaction was allowed to proceedat room temperature for 15 minutes. After completing the reaction, thereaction mixture was adjusted to pH 6.0 with acetic acid to give KiSS-1peptide. This reaction mixture was applied to a Sephadex G-25 column(2.5 cm ID×50 cm L) equilibrated with 50 mM monopotassium phosphate anddevelopment was effected using the same 50 mM monopotassium phosphate asused for equilibration at a flow rate of 10 ml/minute to give KiSS-1peptide fraction. This fraction was further applied to a C4P-50 column(21.5 mm ID 300 mm L, Showa Denko) equilibrated with 0.1%trifluoroacetic acid, for adsorption. After washing the column, stepwisegradient elution was carried out with 20 to 60% B (B: 80%acetonitrile/0.1% trifluoroacetic acid) at a flow rate of 5 mL/minute.The KiSS-1 peptide fractions obtained were pooled and lyophilized togive about 0.35 mg lyophilizate powder of KiSS-1 peptide.

Example 3

Determination of KiSS-1 Peptide Characteristics

a) Amino Acid Composition Analysis

The amino acid composition was determined using an amino acid analyzer(Hitachi model L-8500A amino acid analyzer). As a result, the amino acidcomposition was in agreement with that deduced from the base sequence ofthe DNA for KiSS-1 peptide (Table 1). TABLE 1 Value deduced from Numberof residues base sequence of Amino acid per mole KiSS-1 peptide Asx 3.44 Thr*⁾ 0.9 1 Ser*⁾ 7.1 8 Glx 7.1 7 Pro 8.2 8 Gly 5.1 5 Ala 3.0 3 Cys 00 Val 1.9 2 Met 0 0 Ile 1.0 1 Leu 5 5 Tyr 1.0 1 Phe 1.9 2 His 1.0 1 Lys1.0 1 Arg 3.9 4 Trp 0.4 1Acid hydrolysis (mean value after acid hydrolysis in 6 N HCl-4%thioglycolic acid for 24-48 hrs)*⁾Value obtained by extrapolation to 0 hr.b) N-terminal Amino Acid Sequence Analysis

The N-terminal amino acid sequence was determined using a gaseous phaseprotein sequencer (PE Applied Biosystems model 492). As a result, theN-terminal amino acid sequence was in agreement with that deduced fromthe base sequence of the DNA for KiSS-1 peptide (Table 2). TABLE 2N-terminal Amino Acid Sequence Analysis Amino acid deduced ResiduePTH*⁾-amino acid from base sequence No. Detected of KiSS-1 peptide 1 Gly(56) Gly 2 Thr (52) Thr 3 Ser (41) Ser 4 Leu (45) Leu 5 Ser (33) Ser 6Pro (28) Pro 7 Pro (34) Pro 8 Pro (30) Pro 9 Glu (14) Glu 10 Ser (12)SerAnalysis was made using 100 pmol.*⁾Phenylthiohydantoin.

Example 4

Biological Activity Assay

The activity assay of the KiSS-1 peptide obtained in Example 2 wasperformed by the method described in Example 3 of WO 99/33976(intracellular Ca ion concentration-increasing activity) and it wasconfirmed that its activity was equivalent to that of a sample purifiedfrom human placenta extract.

Example 5

Production of DNA Encoding a KiSS-1 Peptide to Which 24 Amino Acids areAdded at its C-Terminal (2)

pTC2MetC24-1.3, an expression vector for KiSS-1 peptide to which 24amino acids are added at its C-terminal, was constructed using pTC2KiSS1in place of pTFC-KiSS-1, the expression vector for KiSS-1 Peptide usedin Example 1(c) (FIG. 3).

Industrial Applicability

According to the method of the present invention, it is possible toindustrially mass-produce KiSS-1 Peptide or a salt thereof, which can beused as a prophylactic or therapeutic drug for cancers (for example,lung cancer, stomach cancer, liver cancer, pancreatic cancer, colorectalcancer, rectal cancer, colon cancer, prostate cancer, ovarian cancer,uterine cancer, or breast cancer, etc.) as well as for choriocarcinoma,vesicular mole, invasive mole, miscarriage, fetal maldevelopment,saccharometabolic disorder, lipid metabolic disorder, or induction ofdelivery.

1. A method for producing a KiSS-1 peptide or a salt thereof, whichcomprises subjecting a fused protein, a peptide or a salt thereof inwhich a KiSS-1 peptide is ligated to the N-terminal of a low-molecularpeptide having cysteine at its N-terminal to the cleavage reaction ofthe peptide linkage on the amino group side of said cysteine residue. 2.A method for producing a KiSS-1 peptide or a salt thereof, whichcomprises cultivating a transformant having a vector comprising DNAencoding a fused protein or peptide in which a KiSS-1 peptide is ligatedto the N-terminal of a low-molecular peptide having cysteine at itsN-terminal to express the fused protein, the peptide or a salt thereofand subjecting the expressed fused protein, the peptide or the saltthereof to the cleavage reaction of the peptide linkage on the aminogroup side of said cysteine residue.
 3. The method according to claim 1or 2, wherein the C-terminal of the KiSS-1 peptide is an amide.
 4. Themethod according to claim 1 or 2, wherein the cleavage reaction is anS-cyanylation reaction followed by an ammonolysis or a hydrolysis. 5.The method according to claim 1 or 2, wherein the KiSS-1 peptide is apeptide comprising an amino acid sequence of SEQ ID NO:
 1. 6. The methodaccording to claim 1 or 2, wherein the KiSS-1 peptide is (i) a peptidehaving an amino acid sequence of position 40 to position 54 from theN-terminal in the amino acid sequence of SEQ ID NO: 1; (ii) a peptidehaving an amino acid sequence of position 45 to position 54 from theN-terminal in the amino acid sequence of SEQ ID NO: 1; (iii) a peptidehaving an amino acid sequence of position 46 to position 54 from theN-terminal in the amino acid sequence of SEQ ID NO: 1; or (iv) a peptidehaving an amino acid sequence of position 47 to position 54 from theN-terminal in the amino acid sequence of SEQ ID NO:
 1. 7. The methodaccording to claim 1 or 2, wherein the low-molecular peptide havingcysteine at the N-terminal is a peptide consisting of about 10 to about50 amino acid residues.
 8. The method according to claim 1 or 2, whereinthe low-molecular peptide is a partial peptide on the C-terminal side ofa precursor protein comprising the KiSS-1 peptide, and has an amino acidsequence starting from an amino acid residue adjacent to the C-terminalamino acid of the KiSS-1 peptide.
 9. The method according to claim 1 or2, wherein the low-molecular peptide having cysteine at the N-terminalis a peptide that comprises the amino acid sequence of SEQ ID NO: 3 andhas cysteine residue at the N-terminal.
 10. The method according toclaim 1 or 2, wherein the low-molecular peptide having cysteine at theN-terminal is a peptide that comprises an amino acid sequence of SEQ IDNO: 3 and has cysteine residue at the N-terminal; the KiSS-1 peptide isa peptide having an amino acid sequence of SEQ ID NO: 1; and the KiSS-1peptide to be produced is a peptide having an amino acid sequence of SEQID NO: 1 with an amide form of the C-terminal.
 11. A fused protein, apeptide or a salt thereof, in which a KiSS-1 peptide is ligated to theN-terminal of a low-molecular peptide having cysteine at its N-terminal.12. The fused protein, the peptide or the salt thereof according toclaim 11, comprising an amino acid sequence of SEQ ID NO:
 5. 13. DNAcomprising DNA encoding the fused protein or the peptide according toclaim
 11. 14. DNA according to claim 13, having (i) a base sequence ofSEQ ID NO: 6; or (ii) a base sequence of SEQ ID NO:
 7. 15. A vectorcomprising the DNA according to claim
 13. 16. A transformant having thevector according to claim
 15. 17. Escherichia coli MM294(DE3)/pTC2MetC24-1.3 identified as FERM BP-7823.